1. Field of the Invention
The present invention relates to a substantially pure exopolysaccharide adhosin isolated from a particular strain of Staphylococcus epidermidis, to a general method capable of isolating this compound in substantially pure form, and to uses of said purified adhesin product as a vaccine for the production of antibodies effective against the binding of homologous bacterial cells to polymeric materials, and as a probe for the development of polymeric materials useful as catheters and medical prostheses.
2. Description of the Background Art
Both Staphylococcus aureus (coagulase-positive) and Staphylococcus epidermidis (coagulase-negative) have a characteristic propensity for invading skin and adjacent tissues at the site of prosthetic medical devices, including intravascular catheters, cerebrospinal fluid shunts, hemodialysis shunts, vascular grafts, and extended-wear contact lenses. Within 48 to 72 hours, relatively large numbers of staphylococci are demonstrable at the site of insertion of these foreign bodies. Archer, G. L., “Staphylococcus epidermidis: The Organism, Its. Diseases, and Treatment,” in Remington, J. S., et al., eds., Current Clinical Topics in Infectious Diseases, McGraw-Hill, New York, 1986, pp. 25–46; Youmans, G. P., et al., The Biologic and Clinical Basis of Infectious Diseases, Saunders, Philadelphia, 1985, pp. 618–625, 738–9. It has been demonstrated that S. epidermidis cells attach and proliferate on the inner or outer surfaces of catheters, irrespective of their composition (polyethylene, polyvinylchloride, polyvinylfluoride, or polyester based materials).
Although the virulence of coagulase-negative staphylococci clearly is enhanced in the presence of a foreign body, the microbial factor(s) that permit these normal skin commensals to become nosocomial pathogens have not been well characterized. As adherence is believed to be the critical first step in the pathogenesis of coagulase-negative staphylococcal foreign-body infections, attention has focused on surface properties of these organisms that might mediate adherence to, and colonization of, polymeric prosthetic materials.
The most promising candidate for the source of a specific staphylococcal adhesin is an extracellular material often referred to as “slime.” It has been hypothesized that the slime substance may protect the S. epidermidis cells against antibiotics, as well as against natural host defense mechanisms. Youmans et al., supra; Peters, G., et al., Journal of Infectious Diseases 146:479–82 (1982).
It has been known since 1972 that coagulase-negative bacteria isolated from cerebrospinal fluid shunt infections elaborate a mucoid material that stains with alcian blue and is presumably a polysaccharide. Bayston, R., et al., Developmental and Medical Child Neurology 14 (Supp. 27):25–8 (1972). The extracellular polysaccharide substance of slime-producing bacteria is a loose amorphous material composed of a range of low and high molecular weight polymers composed, in gonoral, of neutral monosaccharides such as D-glucose, D-galactose, D-mannose, L-fucose, and L-rhamnose, and also contain amino sugars, uronic acid, and polyols such as ribitol and glycerol. Gristina, A. G., Science 237:1588–95 (1987). Glucose, galactose, phenylalanine, mannose, hexosamine, phosphorous, glycine and alanine have been found as components of the slime produced by S. epidermidis strains in clinical specimens unrelated to biomaterial infections. Ichiman, J., et al., J. Appl. Bact. 51:229 (1981). Isolates of such bacteria from sites of infections are more likely to produce slime than are random isolates from skin. Ishak, M. A., et al., Journal of Clinical Microbiology 22:1025–9 (1985). Moreover, slime-producing strains adhere well to a variety of polymeric materials. Christensen, G. D., et al., Infect. Immun. 37:318–26 (1982).
Coagulase-positive staphylococci (S. aureus) are reported to produce multiple cell surface proteins which can be released from such cells by thermal extraction and which can be shown to bind to influenza virus-infected canine kidney cells. It was considered that S. aureus produces multiple cell surface protein adhesins. Sanford, B. A., et al., Infect. Immun. 52:671–5 (1986); Proc. Soc. Exp. Biol. Med. 181:104–11 (1986).
Identification of other microbial adhesins has been reported. Pier (U.S. Pat. No. 4,285,936, Aug. 25, 1981; U.S. Pat. No. 4,528,458, Mar. 25, 1986) discloses a method for partial purification of a polysaccharide antigen from Pseudomonas aeruginosa slime. Escherichia coli fimbrial protein adhesins have been identified and partially purified by several investigators (Orskov, I., et al., Infect. Immun. 47:191–200 (1985); Chanter, H., J. Gen. Microbiol. 1:225–243 (1983); Ferreiros, C. M., et al., Rev. espanol, de fisiolog, 3:45–50 (1983); and Moch, T., et al., Proc. Natl. Acad. Sci. 84:3462–6 (1987)).
Lectin-like glycoprotein adhesins have been identified in the Bacteroides fragilis group, and a 70 kDa adhesin has been purified by affinity chromatography (Rogemond, V., et al., Infect. Immun. 53:99–102 (1986)). Monoclonal antibody affinity chromatography was used to purify a 165 kDa surface protein of Mycoplasma pneumoniae which mediates attachment of such bacteria to target cells (Leigt, D. K., et al., J. Bacteriol. 157:678–80 (1984)), and to isolate a 150 kDa adhesin protein from Streptococcus sanguis FW213 (Elder, B. L., et al., Infect. Immun. 54:421–7 (1986)). A uroepithelial cell adhesin protein of 17.5 kDa was partially purified from fimbrii of Proteus mirabilis, a frequent cause of urinary tract infection (Wray, S. K., et al., Infect. Immun. 54:43–9 (1986)).
Ludwicka (Ludwicka, A., et al., Zbl. Bakt. Hyg. A 258:256–67 (1984)) fractionated by ion-exchange chromatography a phenol-saline extract of slime from S. epidermidis and obtained four crude fractions. Both the phenol-saline extract and two of the four crude fractions inhibited the attachment of bacterial cells to polymeric material. On the basis of the presence of monosaccharides in the fractions, the reaction of the fractions with lectins, and the complete inhibition of the production of the four fractions by protreatment of the bacteria by tunicamycin (inhibitor of glycoprotein synthesis), the authors concluded that the extracellular slime substance is a complex of glycoconjugate (i.e., glycoprotein) character.
Hogt (Hogt, A. H., et al., Infect, Immun 51:294 (1986) have also observed that crude extracellular products from the slime of homologous strains of S. epidermidis inhibit the adherence of homologous bacterial cells to polymeric materials used as catheters and prostheses. No information was provided in this report as to the chemical nature of the extracellular products.
Bacterial cells and materials derived from the surface of such cells have been used as vaccines to produce antibodies directed against homologous bacteria. Frank (Frank, R., et al., French Patent Application 85-07315, published Nov. 21, 1986) discloses a covalent conjugate between a capsular protein adhesin (MW-74 kDa) from Streptococcus mutans and a polysaccharide from the same (serotypically) organism, and the use of said conjugate as an anti-caries vaccine. Pier (Pier, G. B., et al., U.S. patents, supra) disclose a vaccine comprising a high molecular weight mucoid exopolysaccharide from Pseudomonas aeruginosa strain 2192 slime which induces in recipient animals an immunity to said organism. Sadowski (Sadowski, P., U.S. Pat. No. 4,443,549, Apr. 17, 1984; U.S. Pat. No. 4,652,498, Mar. 24, 1984; and EP 82401506.1, published Apr. 27, 1983) discloses monoclonal antibodies specific for surface adhesions of Escherichia coli and Pseudomonas aeruginosa which may be used for the therapeutic treatment of diseases induced by adhesin-bearing homologous bacteria in humans and animals. Nagy (Nagy, L. K., et al., Dev. Biol. Stand. 53:189–97 (1983)) discloses multi-adhesin vaccines for the protection of the neonatal piglet against Escherichia coli infections.